ADDIS ABABA UNIVERSITY SCHOOL OF GRADUATE STUDIES BIOLOGICAL CONSEQUENCES OF PLEISTOCENE GLACIATIONS IN EAST AFRICAN AFRO-ALPINE ENVIRONMENT AS INFERRED FROM MOLECULAR DATA OF SOME KEY PLANT SPECIES Adane Assefa A Thesis Presented to the School of Graduate Studies of Addis Ababa University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology Addis Ababa, Ethiopia July 2007 ACKNOWLEDGEMENTS I express my sincere gratitude and indebtedness to Dr. Sileshi Nemomissa for accepting me as a PhD student, for his invaluable help in designing and leading my PhD research project as well as providing facilities. I acknowledge and value his competent guidance and unlimited encouragement throughout my study period. The illuminating discussions that I have had with him on many occasions have been very helpful. I am deeply grateful to him for providing helpful suggestions and comments on the manuscripts leading to this dissertation. His thoughtfully recommended me seminar topics that helped me to strengthen my background and that introduced me to the areas of molecular genetics. His ability to facilitate working conditions and sense of understanding has been key factors, when I got stuck at times. His scholar responsibility and kind support did not end there. He successful international collaborations with Professor Christian Brochmann, at National Centre for Biosystematics (NCB), the National History Museum and Botanical Garden, University of Oslo (UIO), Norway and Dr Pierre Taberlet at Université of Joseph Fourier, Grenoble Cedex, France were instrumental in achieving the completion of the work and materializing some of my long-standing ambitions in research. I also owe an enormous debt of gratitude to Professor Christian Brochmann and Dr Pierre Taberlet for their kind willingness to collaborate with Dr Sileshi Nemomissa not only firmly established my admission to PhD studies at AAU under a program known as a “Sandwich Program” but also has given me the opportunity to enjoy the experience and academic atmosphere of the three nations. I am deeply grateful to both of them for their competent and expert supervision, kind support, and the many insightful comments and suggestions that they have had made on the earlier start of the project in field-training on Bale Mountains and on workshops that hold in the Biology department of Addis Ababa University, Ethiopia. I also appreciate Professor Christian’s insight, kind consideration and patience, while holding weekly discussions related to my works in his office for a very long time in the period of my stay in NCB, UIO, Oslo, Norway. This study is fully supported by the Norwegian Programme for Development, Research and High educational (NUFU) Project 54/2003 to Sileshi Nemomissa and Christian Brochmann i (‘AFROALP-Afro-alpine ‘islands’ as natural laboratories: dynamics and units of plant biodiversity’). The School of Graduate Studies (SGS) and Department of Biology, AAU, Ethiopia, and the NCB, National History Museum, UIO, are acknowledged for administering the finances and hosting the study. I deeply acknowledge and appreciate the Education Faculty of Bahir Dar University, (BDU), Bahir Dar, Ethiopia, for providing me study leave with payment as part of its staff development program. I am also grateful to BDU for letting me to get the housing allowance during my study periods. That has given me the feeling that my family members are secured and supported, despite the extended loneliness they had to suffer during the period of my studies. I am indebted to the following institutions/organizations: National Herbarium, AAU for research facilities and support during my field trips; Research and Publication Office (RPO), AAU for facilitating the national and the international field trips, the Biodiversity Conservation and Research Institute, Addis Ababa, Ethiopia for allowing me to take the DNA leaf samples out of the country, the Simen and Bale Mountains National Parks warden offices, Mt Kaka sanctuaries, Mt Choke and Gara Muleta local administrations, University of Dare Es Salaam, Department of Botany, and the Tanzanian Science and Technology Commission for providing me letter of support as well as Kilimanjaro and Arusha National Park warden offices, East Africa National Herbarium and the Kenyan Wildlife Service (KWS), Aberdare National Park, Mt Kenya and Mt Elgon warden offices, for supporting and allowing me to collect DNA leaf specimens and herbarium vouchers. Here, it is appropriate to extend my special thanks to my colleague and the PhD fellowship Ato Mulugeta Kebede for company and help during the whole fieldworks and study periods in and out of the country. I thank also Dr Muasya from East Africa National Herbarium, Kenya, Professor Eric Knox from USA and Dr Berit from Zurich University for company and help during the fieldwork in Kenyan Mountains. Field work was carried out by team of dedicated Rangers and Assistants and I really thank our cooks and drivers in the three countries (Ethiopia, Kenya and Tanzania) without whose efforts this work would not have been possible. Special thanks go to all the porters who assisted me by getting food and equipment to the different campsites of the Simen, Kilimanjaro, and Arusha (Meru) Mountains National Parks. Mrs. Virginia Mierre .patiently taught me DNA extraction and introduced me to the automated thermacycler for the Polymerase Chain Reaction (PCR), as well as to the gel electrophoretic ii materials and to different chemicals. She also committed her time at familiarizing me to the administrative environments of UIO. Mrs. Virginia has not only been extremely helpful for swift acquisition of chemicals and PCR reagents during my laboratory work in NCB, but she also brought me very necessary kitchen equipment at the time of my arrival at Oslo, Norway. Like wise, Dr Dorothee Ehrich cheerfully introduced me to the DNA sequencing technique, AFLP assay, and an automatic genetic analyzer (ABI 3100 PRISM). I sincerely acknowledge and appreciate her pedagogical and friendly approaches, patience and always willingness to help. I am also grateful to Dr. Dorothee for showing me how to use the computer software for data analyses. She devoted her time in prove-reading and commenting on the published papers. I have greatly benefited from the discussions I have had with her at the NCB during the period of my data analysis and the manuscript preparations. Mrs Liv Guro Kvernstuen has been duly acknowledged for rendering invaluable help in the laboratory and data analysis. My appreciation goes to Dr Andreas Tribsch for his help in preliminary screening of the selective PCR primer combinations with Dr Dorothee Ehrich. I also thank Mr Arne Stabel for he has promptly responding to my occasional requests for help when I got a problem running computers, especially in loading and operating Software during the data analysis. I am thankful to W/rt. Genet, Ato Wogie, Ato Solomon, Ato Melaku Wondafrash, W/rt Shewangejiw Lemma and Ato Fiseha for their co-operation and kind help at all the times in the National Herbarium, AAU. I am also thanks to Ato Gezahegn Kassaye and Ato Ijigu from AAU for their driving services throughout the periods of the fieldwork. I acknowledge the library facilities of AAU and the Internet services of the National Herbarium of Addis Ababa University as well as that of the Botanical Garden, NCB, UIO, Norway. Throughout my study period, I have received invaluable help from the M Sc Program students, especially Ato Abel Gizaw and W/rt Birktawit Bekele under the NUFU project and my fellow PhD students, postdoctoral fellowships and researchers in both AAU and NCB. Many times I took some minutes from their busy laboratory or office work to respond thoughtfully to my inquiries and to enlighten my ambiguities. They all have taught me a great deal and their ideas and work have inspired my own. Many thanks to all of them. iii I am thankful to the past and present Heads and Graduate Student Advisors at the Department of Biology, and past and present Deans of SGS, AAU, for facilitating my studies. The secretaries at the Department of Biology, SGS and RPO, AAU, have been instrumental in terms of administrative tasks. Similar roles have been played in the NCB and UIO, Oslo, Norway. They all deserve my special thanks. I wish to thank the finance staff of AAU at Sidist Kilo and Arat Kilo for their services regarding the release of the research money. My very special thanks go to all of my spiritual fathers, mothers, churches and monasteries, spiritual brothers, sisters and Mahibere Kidus Michael and all my friends for cheerful encouragement, considerable help in praying and for taking care of my family throughout my study period. I am deeply grateful especially to my brother Ato Meseret and Ato Seyoum Assefa, and my sisters W/ro Ihetagegn and W/rt Medhanit Assefa, and W/ro Dr Tigist (MD), W/rt Workie Belay, W/rt Firehiwot W/rt Miheret Abera, and W/rt Melkam and my spiritual brothers Ato. Daget Ayana, Obbo Asefaw Regassa, and Ato Ermias Leulkal and his wife W/ro Birtukan, W/ro Mulubirhan Wale and her family for their brotherly and sisterly support and encouragement. I am also appreciatively acknowledge my father-in-law, Ato Abera Belay, my mother-in-law, W/ro Misayie Fentie and Ato Asnake Fentie, and all the rest of brothers and sisters for their encouragement and support for me and for my family throughout my study period. My brother Dr Zerihun Assefa from the Free University of Brussels, Belgium, deserves an enormous debt of gratitude for his fruitful and very constructive criticisms, comments and suggestions on all aspects of my PhD work and he also spent much of his precious time and efforts in critical reading through my seminar papers and dissertation. He also visited me two times from Brussels with his very sweet daughter, Rebecca, and spent several days with me when I was first arrived in Norway, which greatly helped me for adjustment and avoiding the sense of loneliness.